The accessory fibers of the sperm tail

JOURNAL OF ULTRASTRUCTURE RESEARCH 57, 2 8 9 - 3 0 8

(1976)

The Accessory Fibers of the Sperm Tail III. High-Sulfur and Low-Sulfur Components in Mammals and Cephalopods 1 B. BACCETTI, V. PALLINI, AND A. G. B U R R I N I Institute of Zoology, University of Siena, 53100 Siena, Italy Received June 3, 1976 Purified m a m m a l i a n accessory fibers consist of two groups of disulfide-cross-linked polypeptide chains: The high molecular weight chains (42 000-72 000 daltons) are rich in aspartic acid, glutamic acid, and leucine; the low molecular weight chains (28 000-31 000 daltons) are rich in cysteine and proline. The infrared spectrum of whole fibers is similar to t h a t of keratin. Protofibril-like structures, 2 n m thick, are detected in native fibers and become more evident after proteolysis or '~renaturation" from guanidine-HC1 solutions. The cross-striation of accessory fibers originates from the lateral packing of protofibril-like units. Cephalopod accessory fibers are also resolved into two groups of disulfide-cross-linked chains: as in m a m m a l i a n fibers, the high molecular weight chains (about 90 000 daltons in bothEledone moschata and E. cirrhosa) contain large amounts of aspartic acid, glutamic acid, and leucine; the low molecular weight group (about 50 000 and 30 000 daltons in E. moschata, 36 000 daltons in E. cirrhosa) contains large amounts of cysteine, proline, and histidine. The occurrence of low-sulfur and high-sulfur polypeptides, the zinc-binding properties (6), and the analogous localization in wave-generating flagella prompt the authors to distinguish the keratin-like proteins of sperm accessory fibers of m a m m a l s and cephalopods with the new n a m e of parergins.

The accessory fibers of sperm flagella, a flexible system of structures surrounding the actively motile axoneme, have recently been studied both by electron microscopy and by chemical methods after isolation. Electron microscopy demonstrated their occurrence and conspicuous development around the ~9 + 2" basic axoneme in mammals and molluscs, their striated appearance in both of them (2, 3, 5, 17, 35, 36, 51 ), the presence of sulfhydryl groups (32) mostly oxidized to disulfide cross-linkages which stabilize the structure (5, 7, 32), and, finally, the presence of zinc (4, 5, 6, 11-13 ). The chemical approach has revealed in the accessory fibers of bull (5) and rat (3 7) the occurrence of several major polypeptide chains, with molecular weights in a range between 10 000 and >60 000 daltons and distinguishable by high- and low-cysteine chains 1 Research performed u n d e r C.N.R. project '~Biology 0£ reproduction."

(5). In Octopus at least two chains of different molecular weight have been found (3). In addition triglycerides have been demonstrated in purified m a m m a l i a n fibers (5, 37), whereas a high amount of acid-soluble polysaccharide is present in mollusc fiber preparations (6). Phosphates are absent (6). ATPase activity is detectable histochemically (1, 2, 31 ) but not confirmed biochemically on purified fibers (5, 36, 37). More recent data support the hypothesis of an extractable ATPase containing in the matrix of the bull fibers (52) which can be lost during sperm fractionation. In this paper we describe in more detail the protein components of sperm accessory fibers in some m am m al s and cephalopods. The several peculiarities common to these proteins in the two zoological groups prompted us to give t h e m a new name: parergins, from the greek 1r&p~p~o~ = accessory. 289

Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

290

BACCETTI, PALLINI AND BURRINI MATERIALS AND METHODS

(a) Reference proteins and reagents. Bovine ser u m albumin, y-globulin, ovalbumin, cytochrome c, soybean trypsin inhibitor, phenylmethylsulfonylfluoride, and dithiothreitol were Sigma products. The reagents for acrylamide-gel formation and sodium dodecyl sulfate (SDS) were from Serva. Sepharose 4B (Lot No. 7135) was a P h a r m a c i a product. Guanidine-HC1 was a n Erba (Milan) RPE reagent; a 6 M solution h a d an A2so against water of less t h a n 0.02 and was used without further purification. All other chemicals were reagent grade products. Myosin was purified from rabbit skeletal muscle following the method of Nauss et al. (30). Rabbit skeletal muscle actin was prepared following the method of Drabikowsky and Gergely (15) under the conditions which do not completely remove tropomyosin; the latter protein was therefore present in the preparations of actin. Sea urchin sperm axonemes prepared after Shelanski and Taylor (42) were used as source of tubulin. [14C]Iodoacetamide (Batch 28) was a product of the Radiochemical Center, Amersham. (b) Preparations of the accessory fibers. Spermatozoa from m a m m a l i a n and cephalopod species were collected as previously described (6). Accesory fibers were purified by density-step centrifugation of sonicated sperm (6). Some purifications were performed by including soybean trypsin inhibitor (1 mg/ml) or phenylmethylsulfonylfluoride (0.3 mg/ml) in the NaC1 and sucrose solutions. (c) Reduction and carboxarnidomethylation. Purified accessory fibers were precipitated with 5% trichloroacetic acid in the cold and centrifuged at 30 000g for 10 min, the sediment was washed with ether and dried in vacuo. These t r e a t m e n t s remove bound metals, the acid-soluble polysaccharide present in cephalopod fiber preparations, and the triglyceride-like substances associated with m a m m a lian fibers (5, 37). Reduction of disulfide bridges was performed at a fiber concentration of 4-6 mg dry weight/ml in 7 M guanidine-HC1 buffered at pH 8.5 with 0.5 M Tris-HC1, in the presence of 0.1 M dithiothreitol for 2 h r at 37°C u n d e r nitrogen. Carboxamidomethylation of sulfhydryl groups was achieved by adding iodoacetamide to a final concentration of 0.25 M. Fiber proteins were recovered from reaction mixtures by precipitation w i t h 5 vol of absolute ethanol at - 20°C and centrifugation at 5000g at 2°C, washed with cold ethanol, and dried in vacuo. Alternatively, the reduced alkylated proteins could be precipitated by dialysis of the reaction mixtures against excess volumes of distilled water (with at least two changes) at room t e m p e r a t u r e and recovered by centrifugation as described above. (d) SDS-polyacrylamide electrophoresis. The high pH discontinuous system containing 0.1% SDS described by Laemmli (26) was used, with 5.5 x 55-mm

cylindrical separation gels of 10 or 12% polyacrylamide. Reference and fiber proteins were dissolved by heating at 100°C for 2 min in Laemmli's ~¢final sample buffer" (25) consisting of 2% SDS, 5% mercaptoethanol, 0.06 M Tris-HC1 o f p H 6.8, 10% glycerol, and 0.001% bromphenol blue. Electrophoresis was performed at 1 mA/gel. Protein bands were stained for 1 h r with 0.2% Coomassie blue in 50% methanol containing 7% acetic acid for 1 h r and destained following the method of Maizel (28). The PAS reaction for glycoproteins was performed after F a i r b a n k s et al. (16). The relative mobility of the bands was calculated as the ratio between t h e i r migration and the migration of the ~buffer discontin u i t y ' (28) which corresponded to the upper part of the tracking dye band and retained the same stain in our experimental conditions. A p p a r e n t molecular weights were calculated by interpolation into calibration lines obtained with proteins of known molecular weight r u n in the same electrophoretic experiment. The following reference proteins have been used: myosin heavy chains (MW: 200 000), bovine serum a l b u m i n (MW: 68 000), ~/-globulin h e a v y chain (MW: 50 000), ~/-globulin light chain (MW: 23 500), actin (MW: 45 000), tropomyosin (MW: 36 000), carboxymethylated papain (MW: 23 000), and cytochrome c (MW: 11 700). Tubulin was separated into components 1 and 2 (MW: 56 000 and 53 000 (50)) on 10% polyacrylamide gels; on 12% gels tubulin migrated as a single band, and a n average molecular weight of 54,500 was used. The migration of myosin heavy chain did not fit into the calibration lines, as has been observed by other authors (28); therefore the assignments of molecular weight values above 68 000 (serum albumin) are to be regarded as tentative. In order to estimate protein content, stained electrophoretic bands were cut, rinsed with distilled water, and incubated at 40°C with 25% aqueous pyridine until all the dye was eluted, and the A~o5 of the solution was measured (18). We have observed t h a t this procedure does not remove any protein from the gels, since it allows a perfect restaining and does not extract radioactivity from labeled bands. Therefore, radioactivity measurements on labeled bands could be performed by the method of Semor (41) after protein quantification by m e a s u r e m e n t of absorbance of the eluted dye and removal of pyridine from the gel sections by rinsing with water. (e) Gel filtration. A preparative separation of carboxamidomethylated accessory fiber proteins was performed on a 1.6 x 85-cm column of Sepharose 4B equilibrated and eluted with 6 M guanidine-HC1 following the indications o f F i s h et al. (19). Fractions of about 1 g were collected with a drop-counting fraction collector at a flow rate of about 1 g/48 min. The weight (19) and A~s0 of each fraction were determined. The void volume (V0) of the column was measured from the elution volume (apex of the

PARERGINS: PROTEINS OF SPERM ACCESSORY FIBERS peak) of blue dextran 2000. For column calibration, the following reference proteins were used after reduction and carboxamidomethylation as described above: myosin heavy chain (MW: 200 000), bovine serum a l b u m i n (MW: 68 000), ovalbumin (MW: 43 000), p a p a i n (MW: 23 700), and cytochrome c (MW: 11 700). The ratio between the elution volume (Ve) and the void volume of the column (Vo) was determined for each reference protein and plotted against the logarithm of the molecular weight (19). The resulting calibration line allowed the determin a t i o n of the a p p a r e n t molecular weight of accessory fiber polypeptides. The Ve/Vo for myosin h e a v y chain did not fit the calibration line, as observed by other authors (19) for h i g h molecular weight polypeptides; therefore, the determinations of molecular weight above 68 000 (bovine serum albumin) are to be regarded as tentative. (f) Amino acid analysis. Unmodified or reduced carboxamidomethylated fiber proteins were hydrolyzed for 24 h r a t 110°C following the method of Moore and Stein (29). The hydrolysates were dried in vacuo over P20~ and NaOH and analyzed with the M 82 single column of a Beckman Multichrom B liquid column chromatograph. With alkylated material, a first buffer of slightly reduced pH (3.17 instead of 3.22) was used in order to achieve a good separation of S-carboxymethylcysteine from aspartic acid. No corrections were made for amino acid decomposition during hydrolysis. (g) Infrared spectroscopy. Thin films of bull and Eledone moschata accessory fibers were prepared by stroking drops of dense water suspensions to dryness on I n t r a n windows and were t h e n dried in vacuo over P20.~. The films were not birefringent and the recorded spectra did not result in being dichroic. Spectra were recorded with a P e r k i n - E l m e r 225 grating infrared spectrophotometer. (h) Electron microscopy. Ejaculated h u m a n spermatozoa were fixed in 2.5% glutaraldehyde in cacodylate buffer for i hr, washed, postfixed in 1% O s Q for 30 min, dehydrated, and embedded in Epon. Isolated accessory fibers from m a m m a l i a n and cephalopod sperm were negatively stained with 2% PTA, u r a n y l acetate, and u r a n y l formate on Formvat-coated copper grids. The specimens were examined in a Philips EM 301 electron microscope operated at 60 kV. RESULTS

Chemical Properties of Whole Fiber Proteins Purified fibers from mammalian and cephalopod sperm showed very similar solubility properties. In fact, they did not dissolve in 0.1 N HC1 at room temperature or in 1% SDS, 8 M urea, 7 M guanidineHC1 (all solutions buffered at pH 8.5 with

291

0.5 M Tris-HC1) at room temperature or 100°C. A swelling and eventually an incomplete solubilization of the fibers with the formation of a viscous opalescent gel were obtained in 1 N NaOH. Some swelling was also produced by concentrated dioxane or dimethyl formamide. A complete solubilization was promptly achieved at room temperature in 1% SDS or 7 M guanidine-HC1 (solutions buffered at pH 8.5 with 0.5 M Tris-HC1) in the presence of 0.1 M mercaptoethanol or dithiothreitol. The reduced carboxyamidomethylated whole fiber proteins of mammals and cephalopods (cf. Materials and Methods) were readily dissolved by 6 M guanidine-HC1 at room temperature or 1% SDS at 100°C; unlike mammalian tiber proteins, the cephalopod proteins did not dissolve in 8 M urea or 0.1 M NaOH after carboxamidomethylation. In 6 M guanidine-HC1 an absorbance at 280 nm of 1 unit/mg dry weight/ml with 1 1-cm light path was observed for alkylated whole fiber protein from bull, Eledone moschata, and E. cirrhosa sperm. The ratio A28o/A26o was about 1.3 in bull fiber and 0.7-0.8 in the Eledone moschata and E. cirrhosa fiber protein. The amino acid composition of whole fibers of three mammalian species (Table I) shows, as a common feature, a high content of aspartic acid, glutamic acid, leucine, and serine; a low content oftyrosine, phenylalanine, histidine, and methionine; cysteine and proline are present in large amounts, more markedly in bull and rat than in h u m a n fibers. The most striking character in the amino acid composition of mollusc fibers is the high content of histidine (Table I); like mammalian fibers, cephalopod fibers are rich in cysteine and proline and poor in tyrosine, phenylalanine, and methionine. In both maremals and cephalopods, polar amino acids predominate over nonpolar ones. The low degree of variability in the amino acid composition in several preparations of bull and Eledone moschata fibers indicates that the purification proce-

292

BACCETTI, P A L L I N I A N D B U R R I N I TABLE I

AMINO ACID COMPOSITION OF MAMMALIAN AND CEPHALOPOD WHOLE FIBERS (RESIDUES/100 RESIDUES) A m i n o acid Alanine Arginine A s p a r t i c acid e Cysteine G l u t a m i c acid ~ Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine

Bos taurus a

5.27 6.48 9.93 7.44 10.46 4.98 1.67 3.20 9.97 6.61 1.90 2.18 8.58 9.22 3.82 3.54 4.81

_+ 0.43 _+ 0.59 _+ 0.85 _+ 0.86 _+ 0.43 _+ 0.42 _+ 0.11 -- 0.31 _+ 0.55 _+ 0.54 _+ 0.23 _+ 0.28 _+ 0.54 _+ 0.43 _+ 0.27 _+ 0.33 _+ 0.43

Rattus rattus b

Homo sapiens b

4.96 5.62 8.99 7.98 9.84 7.46 1.54 3.10 9.29 6.84 1.58 2.00 8.81 8.87 4.42 3.76 4.97

6.64 5.34 9.96 5.34 12.94 6.78 2.37 3.41 8.79 6.56 1.65 2.79 5.74 8.90 4.98 3.02 4.85

Eledone moschata c

5.92 3.40 5.68 6.17 7.85 8.82 14.88 4.15 4.20 6.27 1.57 1.83 10.75 6.05 5.24 2.24 5.00

- 0.59 -+ 0.35 _+ 0.40 -+ 0.59 -+ 0.60 _+ 0.57 _+ 1.73 _+ 0.30 _+ 0.29 -+ 0.59 -+ 0.14 _+ 0.12 _+ 0.90 -+ 0.50 _+ 0.19 -+ 0.23 -+ 0.42

E. cirrhosad

Octopus v ulgaris d

6.01 4.11 5.72 9.56 7.85 7.70 12.97 3.34 4.02 6.04 1.09 1.31 11.99 5.75 4.90 2.98 4.67

6.14 8.06 7.00 7.65 6.50 9.72 10.45 3.88 4.03 7.06 0.81 1.71 9.68 5.54 5.29 1.82 4.66

a A v e r a g e of a n a l y s e s on six different fiber p r e p a r a t i o n s -+ s t a n d a r d deviation. In four a n a l y s e s cysteine w a s d e t e r m i n e d as 1/2-cystine; in two a n a l y s e s cysteine w a s d e t e r m i n e d as S - c a r b o x y m e t h y l c y s t e i n e . A v e r a g e of a n a l y s e s on two different p r e p a r a t i o n s . Cysteine w a s d e t e r m i n e d as S - c a r b o x y m e t h y l c y s teine. c A v e r a g e of a n a l y s e s on five different p r e p a r a t i o n s _+ s t a n d a r d deviation. Cysteine w a s d e t e r m i n e d as 1/2-cystine. d A v e r a g e of a n a l y s e s on two different p r e p a r a t i o n s . Cysteine w a s d e t e r m i n e d as 1/2-cystine. e Includes a s p a r t i c acid and a s p a r a g i n e . Includes g l u t a m i c acid and g l u t a m i n e .

dure yields reproducible results. The standard deviations reported for bull and Eledone moschata fibers are slightly higher than 10% in only a few cases. The accelerated method of amino acid analysis (cf. Materials and Methods) has itself a precision of about 5%.

Polypeptide Chain Composition Bull, rat, and human accessory fibers consist of polypeptide chains of quite similar number and molecular weight, as shown in Fig. 1. Upon electrophoresis in 10% polyacrylamide gels the following principal bands are noted: (a) a sharp band of relative mobility 0.23 in bull, 0.24 in rat, and 0.22 in h u m a n fibers, which corresponds to a polypeptide chain of 72 00075 000 daltons; (b) a band (two very close sharp bands in some bull fiber preparations) of relative mobility 0.33 in bull, 0.34 in rat, and 0.35 in h u m a n fibers, corresponding to a molecular weight of 54 000-

57 000; (c) a main intense band, with a diffuse trailing edge, of relative mobility 0.58 in bull and 0.61 in rat fibers, corresponding to molecular weights of 31 000 and 29 000, respectively. Details not clearly visible in the picture indicate that this intense band consists of at least two closely migrating bands; in human fibers, in fact, two clearly separated intense bands are visible which correspond to relative mobilities of 0.58 and 0.62 and to molecular weights of 31 000 and 28 000. (d) There is a relatively faint band of relative mobility 0.69 (MW: about 24 000), parallel to bands of similar mobility in rat and human fibers. (e) The lower portion of the gels shows minor bands and some stained material migrating at the buffer discontinuity in all species studied. Minor bands were occasionally present in mammalian fiber preparations. The principal components with molecular weights ranging between 72 000 and

293

PARERGINS: PROTEINS OF SPERM ACCESSORY FIBERS 1.0

ii I i

.24

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.35

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Fro. 1. Electrophoresis on 12% SDS-polyacrylamide of reduced carboxamidomethylated m a m m a l i a n accessory fiber proteins. (a) Bull fibers, 100 t~g; (b) r a t fibers, 90 t'g; (c) h u m a n fibers, 70 t*g. The relative mobility of the b a n d s is indicated with nnmbers, and the arrows m a r k the buffer discontinuity. The calibration line was obtained with the following reference proteins: (a) cytochrome c; (b) papain; (c) ~/globulin light chain; (d) tropomyosin; (e) actin; (f) T-globulin heavy chain; (g) tubulin; (h) bovine serum albumin; (i) myosin heavy chain.

75 000, 54 000 and 57 000, 29 000 and 31 000 (28 000 and 31 000 in human fibers), as well as the material migrating at the buffer discontinuity were observed in various preparations (eight of bull, two of rat and h u m a n fibers) analyzed by SDSpolyacrylamide electrophoresis. The relarive intensities of these bands, measured from the absorbance of the eluted dye, were also nearly constant in several preparations; the values are reported in Table II. The components with molecular weights between 29 000 and 30 000 were by far predominant over the higher molecular weight polypeptides, particularly in bull and rat fibers. In h u m a n fibers, the high molecular weight components (about 80 000 and 55 000 daltons) accounted for a higher percentage of total dye than in rat and bull fibers; of the two closely migrating chains (MW: 28 000 and 31 000), the lighter one was the more intense one. It is also interesting to note that none of the described bands (Figs. 1 and 2) corresponds to the

TABLE II INTENSITY OF ELECTROPHORETIC BANDS FROM MAMMALIAN ACC'ESSORY FIBERS

Band (relative mobility)

Percentage of total dye

Bos taurus a

18_+4 10_+5 56 -+ 10 16-+8

0.23 0.33

0.58 Other bands

Rattus rattus ~

0.24

0.34 0.61 Other bands

16 16 47 21

15 17 44 24

28 24 16 17 15

23 23 17 14 23

Homo sapiens b

0.22 0.35 0.bS 0.62 Other bands

a Data from eight preparations x standard deviation. b Data from two preparations.

migration of myosin heavy chains (relative mobility about 0.1) or of actin (relative mobility 0.43).

294

BACCETTI, PALLINI AND BURRINI

1.0, .g,

~i!~

:i

~0.29 ~0.30 0,49

.8. 0.29 0.30

i

~

O.4 9 0.55

--0,44 ....- - 0 . 4 7

wo.61

0.67

.•0,67

.7 ,6

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i !!ii

M . W . x 10 -~ FIG. 2. Electrophoresis on 10% SDS-polyacrylamide of reduced carboxamidomethylated cephalopod accessory fiber proteins. (a) Eledone moschata fibers, 120 /~g; (b) Octopus vulgaris fibers, 80/Lg; (c) Eledone cirrhosa fibers, 80/~g. The relative mobility of the bands is indicated with numbers, and the arrows mark the buffer discontinuity. The calibration line was obtained with the following reference proteins: (a) cytochrome c; (b)papain; (c)~-globulin light chain; (d)tropomyosin; (e)actin; 05 ~/-globulin heavy chain; (g) tubulin 2; (g) tubulin 1; (h) bovine serum albumin; (i) myosin heavy chain.

Unlike mammalian fibers, cephalopod fibers consist of polypeptide chains almost species specific as to number and molecular weight (Fig. 2). The only correspondence between Eledone moschata and E. cirrhosa is represented by the two closely migrating bands with relative mobilities (on 12% polyacrylamide gels) of 0.29 and 0.30 (MW: about 90 000-94 000). In addition, Eledone moschata fibers showed two intense bands at relative mobilities of 0.49 (sometimes resolved into a group of bands) and 0.67 (MW: 53 000 and 31 000), whereas Eledone cirrhosa fibers had only one intense band of relative mobility 0.61 (MW: about 36 000); constant characteristics of the Eledone cirrhosa fibers were also two thin bands with relative mobilities of 0.44 and 0.47. The predominant polypeptides present in Octopus fibers showed relative mobilities of 0.49 and 0.55 (MW: 53 000 and 44 000). The relative intensities of cephalopod fiber bands are reported in Table III. None of the electropho-

TABLE III INTENSITY OF ELECTROPHORETIC BANDS FROM CEPHALOPOD ACCESSORY FIBERS

Band (relative mobility)

Percentage of total dye

Eledone moschata ~

0.29 + 0.30 0.49 0.67 Other bands

16 24 22 38

14 26 18 42

Eledone cirrhosa b

0,29 + 0.30 0.44 + 0.47 0.60 Other bands

25 20 30 25

Octopus vulgaris b

0.49 0.55 Other bands

35 44 21

a Data from two preparations. b Data from one preparation.

retic bands observed in mammal and cephalopod fiber preparations reacted positively when the gels were stained for glycoproteins. Some reacting material was observed on top of "spacer" and "separa-

295

PARERGINS: PROTEINS OF SPERM ACCESSORY FIBERS

fiber preparations with quite similar relative intensities (Tables II and III). They were not present in the fractions containing nuclei (6). They were present with relatively very low intensity together with m a n y other bands in the fractions containing microsomes and microtubules, where small fragments of accessory fibers were also detected by electron microscopy (6). Inclusion of proteolytic enzyme inhibitors (soybean trypsin inhibitor or phenylmethylsulfonylfluoride) in the sperm collection and fractionation procedures, did not alter the electrophoretic pattern of the fiber preparations.

tion" gels when cephalopod fibers were electrophoresed. The above described main electrophoretic bands may be confidently attributed to the polypeptide chains constituting the accessory fibers on the basis of the electron microscopic purity of the preparations. Moreover, the main bands observed in Figs. i and 2 were present in several 200

a--%

\ \ \~\~1,32 ~1,33

100 80 60

1~50 ~. c--o

40

1,55 ~1,66

:S

__ 20

Amino Acid Composition of Mammalian Fiber Polypeptides A separation on a preparative scale of the polypeptide chains present in the accessory fibers was attempted by gel filtration on Sepharose 4B equilibrated and eluted with 6 M guanidine-HC1. The results of the calibration of the Sepharose column with reference proteins are reported in Fig. 3. Figure 4 shows a typical chromatogram

1,86 •

I0 @

110

'

1;2

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1;4

'

116

1,8

2,0

2,2

ve/Vo

FIG. 3. Calibration of the Sepharose 4B column for the d e t e r m i n a t i o n of molecular weight. (a) Myosin heavy chain, (b) bovine serum albumin, (c) ovalbumin, (d) papain, (e) cytochrome c. The numbers on the r i g h t side of the line indicate the VJVo ratios from bull, Eledone moschata, and Eledone cirrhosa chromatograms (Figs• 4 and 5).

'

i

.1

i

(~)

1,0

i

i

1,2

i

1,4

i

1,6

i

i

1,8

i

2,0

2,2

Ve/Vo FIG. 4. Gel filtration on Sepharose 4B of bull accessory fiber proteins. The column (cf. Materials and Methods) was loaded with 15 mg (15 A2s0 units) of reduced carboxamidomethylated protein in 0.3 ml of 6 M guanidine-HC1. The position of the fractions in the chromatogram is indicated as VJVo ratio (abscissa); the protein concentration in the fractions is indicated by the A ~8ovalue (ordinate). The recovery of A 2sofrom the column was 92%. Fractions in the dashed areas were pooled, and the protein was precipitated with 5 vol of absolute ethanol. A m o u n t s of protein corresponding to 0.03-0• 10A~s0 u n i t were electrophoresed on 12% SDSpolyacrylamide (insets, gels; the gel on the left side of the chromatogram was obtained with 0.15 A 2s0 unit of alkylated whole fiber proteins). Amounts of similarly precipitated protein corresponding to 0.2-0.3 A28o u n i t were hydrolyzed for amino acid analyses (reported in Table IV).

296

BACCETTI, PALLINI AND BURRINI

of reduced carboxamidomethylated bull fiber proteins. The content of the chromatographic fractions corresponding to the dashed areas was analyzed by electrophoresis on 12% polyacrylamide. The main features may be described as follows: (a) There is a small peak at the void volume of the column (Ve/Vo = 1). (b) There is a shoulder at a Ve/Vo of 1.50 corresponding to a molecular weight (cf. Fig. 3) of about 55 000. It contains mainly the polypeptide chain whose molecular weight was determined to be about 75 000 by electrophoresis on 12% polyacrylamide (relative mobility of 0.23 in Fig. 1). The present data do not allow any explanation for this discrepancy; as a matter of fact, this polypeptide behaved heavier in electrophoresis and lighter in get filtration than reference bovine serum albumin. (c) A peak at a Ve/Vo of 1.65 corresponds to a molecular weight (cf. Fig. 3) of about 42 000. By electrophoresis on 12% polyacrylamide it is shown to consist mainly of the two close bands migrating with an average molecular weight of about 55 000 (relative mobility of about 0.33 in Fig. 1). This component, too, migrated differently in gel filtration and electrophoresis with respect to reference ovalbumin and actin. (d) A peak with a V J V o of 1.85 corresponds to a molecular weight of about 29 000 (Fig. 3); it contains the intense electrophoretic band with diffuse trailing edge characterized by a relative mobility of 0.58 and by a molecular weight of about 31 000 (Fig. 1). (e) There is a shoulder at a Ve/Vo of about 1.98; the fractions in this area contain in approximately equal amounts the electrophoretic bands with relative mobilities of 0.58 and 0.69. (i) A peak at a V J V o of 2.21 contains a material which is electrophoresed at the buffer discontinuity on 12% polyacrylamide gels. The material forming the shoulder at Ve/Vo = 1.50, the peak at Ve/Vo = 1.66, and the peak at Ve/Vo = 1.85 (Fig. 4) consisted of electrophoretically quite pure polypeptide chains and was recovered in sufficient quantity for amino acid analysis. The

results are reported in Table IV. The higher molecular weight polypeptides contain large amounts of aspartic acid, glutamic acid, and leucine and relatively little carboxymethylcysteine and proline; the 29 000 to 30 000-dalton polypeptide, on the contrary, contains relatively much more proline and carboxymethylcysteine and relatively less glutamic acid and aspartic acid; these three polypeptides do not show striking differences in the content of the other amino acids. No traces of 1/2-cystine were found in the amino acid chromatograms of the polypeptide chains; this fact indicates that a complete reduction and alkylation had been achieved. The limited availability of rat and human semen prevented a preparative separation of the fiber polypeptide chains and the determination of their amino acid compositions. In order to establish whether a high-cysteine polypeptide with a molecular weight of 28 000-30 000, similar to that found in bull fibers, is also present in other mammalian species, rat and h u m a n fibers were reduced in 7 M guanidine-HC1 and alkylated with ['4C]iodoacetamide; the distribution of radioactivity among the polypeptide chains separated by SDS-acrylamide electrophoresis was measured and referred to the dye intensity of the bands. Since radioactive iodoacetamide labels essentially cysteine under the alkylation conditions described (22) (cf. Materials and Methods) and the absorbance of the eluted dye is related to the protein content of the band, the ratio of counts per minute (cpm) per unit of A605 is a measure of the cysteine content of the polypeptide Chains. This procedure was applied to rat and human fibers and for comparison to two different bull fiber preparations. The results are reported in Table V. The intense bands characterized by relative mobilities of 0.58 in bull, 0.61 in rat, and 0.62 in human fibers (cf. Fig. 1), which result in being the most labeled ones, are to be considered as the most cysteine rich. These results agree with the amino acid composition of bull

297

PARERGINS: PROTEINS OF SPERM ACCESSORY FIBERS TABLE IV AMINO ACID COMPOSITION OF BULL POLYPEPTIDE CHAINS (RESIDUES/100 RESIDUES) Ve/Vo ~ MW b MW c

Amino acid Alanine Arginine Aspartic acid ~ Cysteine e G l u t a m i c acid ~ Glycine Histidine Isoleucine Leucine Lysine Methionine Phenyl alanine Proline Serine Threonine Tyrosine Valine

1.45-1.54 55 000 72 000

1.61-1.68 42 000 55 000

1.80-1.86 29 000 31 000

SCMKA ~

SCMKB h

6.38 6.17 10.43 4.01 12.64 4.48 1.76 3.27 9.55 8.22 3.00 2.35 5.81 9.64 4.69 2.70 4.97

6.80 5.93 10.33 4.01 14.38 4.62 1.66 3.08 9.86 9.23 3.03 1.92 5.02 8.75 4.08 2.38 4.91

3.73 7.17 8.05 15.90 6.07 4.40 1.18 3.45 7.07 5.94 1.35 1.77 12.09 9.60 2.35 4.72 5.24

6.4 7.3 8.1 6.8 14.1 8.8 0.7 3.7 10.3 4.1 0.6 3.0 4.2 7.3 4.4 4.3 5.9

2.9 6.7 4.1 17.9 6.4 5.4 0.9 3.0 5.0 0.7 0.0 2.4 13.6 12.0 10.4 1.9 6.7

a Indicates the Ve/Vo r a n g e of the pooled fractions from gel filtration (cf. Fig. 4). b Molecular weight from gel filtration (cf. Figs. 3 and 4). ~ Molecular weight from SDS-polyacrylamide electrophoresis (cf. Fig. 1). d Includes aspartic acid and asparagine. e Determined as S-carboxymethylcysteine, Includes g l u t a m i c acid and g l u t a m i n e . K e r a t i n low-sulfur protein, d a t a from Crewther et al. (1965) quoted in Ref. (9). h K e r a t i n h i g h - s u l f u r protein, d a t a from Lindley et al. (1971) quoted in Ref. (9).

polypeptide chains (cf. Table IV) determined after gel filtration. These bands from bull and rat fibers show relatively little radioactivity at the level of the trailing edge; this region of the gels, therefore, probably contains a polypeptide chain of a lower cysteine content, different from that constituting the main portion of the band. In this connection it is interesting to note that the band of relative mobility 0.58 from human fibers is also less radioactive than the closely migrating band of relative mobility 0.62. In conclusion, these results indicate that bull, rat, and h u m a n fibers possess a polypeptide chain characterized by a high cysteine content, of rather similar molecular weight (31 000 in bull, 29 000 in rat, and 28 000 in human fibers). This chain migrates on electrophoresis quite closely to a different polypeptide of higher molecular weight and of lower cys-

teine content. These two chains are well separated in human fibers, where they show a relative mobility of 0.58 and 0.61; in bull and rat fibers the two chains are not well separated and the low-cysteine chain constitutes the trailing edge of a main band. These chains are conceivably eluted together in the gel-filtration peak of VJVo = 1.85 from bull fibers (cf. Fig. 4) where high percentages of cysteine and proline and also aspartic acid, glutamic acid, and leucine are present (cf. Table IV).

Amino Acid Composition of Cephalopod Fiber Polypeptides A typical chromatogram on Sepharose 4B of reduced carboxamidomethylated protein from Eledone cirrhosa fibers is shown in Fig. 5. Its features may be described as follows: (a) A conspicuous peak is eluted at the void volume of the column (Ve/Vo = 1);

298

BACCETTI, PALLINI AND BURRINI

TABLE V DISTRIBUTION OF [14C]IoDOACETAMIDE IN ELECTROPHORETIC BANDS FROM MAMMALIAN ACCESSORY FIBERSa Electrophoretic bands Degree of labeling b (relative mobility) (cpm/A 6o5) Bog t a u r u s c

0.23 0.33 0.58 (trailing edge) 0.58

383 340 227 1382

360 395 215 1387

Rattus rattus d

0.24 0.34 0.61 (trailing edge) 0.61

373 339 447 1471

Homo sapiens ~

0.22 0.35 0.58 0.62

316 337 307 1327

a Accessory fiber proteins were reduced and alkylated (Cf. Materials and Methods) w i t h [14C]Iodoacetamide (sp act: 100 t~Ci/mmole). About 100 tLg of labeled protein were electrophoresed on 10% SDS-polyacrylamide gels, and the stained bands were counted after elution of the dye (cf. Materials and Methods). b Expressed as the ratio between counts per minute and the absorbance of the eluted dye. c Data from two fiber preparations. d Data from one fiber preparation.

it contains a material which does not enter the gel when analyzed on 10% polyacrylamide. (b) A second peak, eluted at a V J Vo of 1.33, corresponds to a molecular weight of approximately 90 000 (Fig. 3); it consists of the two polypeptide chains which show, on electrophoresis, a molecular weight of about 90 000 (relative mobilities 0.29-0.30 in Fig. 2); this peak also contains some material which barely enters the acrylamide gel. (c) The shoulder at a Ve/Vo of 1.5 corresponds to the two faint electrophoretic bands (relative mobilities of 0.44 and 0.47 in Fig. 2) which have been described above as characteristic of Eledone cirrhosa fibers. (d) There is a peak with a Ve/Vo of 1.7 (MW: 38 000) which consists mainly of the 36 000-dalton electrophoretic band (relative mobility of 0.61 in Fig. 2) and, to a minor extent, of faster-

migrating material. (e) There is a small peak with a Ve/Vo of about 2.15; it consists of material migrating at the buffer boundary.

The proteins from Eledone moschata fibers are eluted from the Sepharose 4B colu m n as shown in Fig. 5. The following principal characteristics are described: (a) A peak of considerable size elutes at the void volume of the column (Ve/Vo = 1); its components do not enter 10% polyacrylamide gels. (b) The peak at Ve/Vo = 1.32 corresponds to a molecular weight of 95 000-100 000 (Fig. 3) and consists of the two closely spaced polypeptide chains which on 10% polyacrylamide show a molecular weight of 90 000-94 000 (relative mobilities 0.29-0.30 in Fig. 2). (c) The peak at V J V o = 1.55 corresponds to a molecular weight of about 50 000 (Fig. 3) and is shown to belong to the group of polypeptide chains whose main component shows, electrophoretically, a molecular weight of 53,000 (relative mobility = 0.49 in Fig. 2). (d) The fourth peak (Ve/Vo = 1.86) corresponds to a molecular weight of about 28 000 (Fig. 3). It consists mainly of the polypeptide chain whose electrophoretic molecular weight is about 31 000 (relative mobility = 0.67 in Fig. 2). (e) Two poorly resolved minor peaks elute at V J V o values of 2.16 and 2.23; they contain material which migrates to the buffer boundary on 10% polyacrylamide gels. The amino acid compositions of the material forming the peaks with V JV o ratios of 1, 1.33, and 1.7 from E. cirrhosa and with Ve/Vo ratios of 1, 1.32, 1.55, and 1.86 from E. moschata are reported in Table VI. It is clearly noted that in both species the high molecular weight components are relatively rich in aspartic acid, glutamic acid, and leucine and relatively poor in cysteine and proline. The opposite situation is found in the lower molecular weight polypeptide chains. An analogous distribution of these amino acids had been observed in the polypeptide chains of bull accessory fibers (cf. Table IV). Histidine,

299

PARERGINS: PROTEINS OF SPERM ACCESSORY FIBERS

.4

.2

C Ol E

o

t~

E.moschata~

f

/I

(rt

Q

~

1,0

1,2

-

1,4

1,6 Ve/Vo

1

1,8

2,0

2,2

....

FIG. 5. Gel filtration on Sepharose 4B of Eledone cirrhosa and Eledone moschata accessory fiber )roteins. The column (cf. Materials and Methods) was loaded with 30 m g (30 A2so units) of reduced arboxamidomethylated protein in 1 ml of 6 M guanidine-HC1. The position of the fractions in the h r o m a t o g r a m is indicated as V~/Vo ratio (abscissa); the protein concentration in the fractions is indicated ,y the A2so value (ordinate). The recovery of A2so from the column was about 80%. Fractions in the dashed .reas were pooled, and the protein was precipitated with 5 vol of absolute ethanol. A m o u n t s of protein orresponding t¢} 0.03-0.10A~8o u n i t s were electrophoresed on 10% SDS-polyacrylamide (insets, gels; the gels n the r i g h t side of the chromatogram were obtained with 0.15 A~so u n i t of alkylated whole fiber proteins). ~mounts of similarly precipitated protein corresponding to 0.2-0.3 A28o unit were hydrolyzed for amino acid nalyses (reported in Table VI).

lery abundant in Eledone fibers, is found )redominantly in the low molecular veight components together with cysteine ,nd proline. Intermediate characteristics Lre found in the peak from E. moschata ibers eluted at a Ve/Vo of 1.55; this peak, n fact, is shown to contain a group of ,olypeptides that migrate very closely on lectrophoresis (Fig. 5). It is also to be oted that in both Eledone species the ma~rial eluted at the void volume of the olumn has an amino acid composition imilar to that found in the nearest re~rded peak (VJVo of 1.83 in E. cirrhosa nd of 1.81 in E. moschata). This fact sugests that the polypeptide chains present the latter peaks may form higher moleclar weight aggregates; similar aggreates may occur in SDS solutions and rep-

resent the material which does not enter the gel when the high molecular chains purified by gel filtration are electrophoresed on 10% polyacrylamide (Fig. 5). This association is most probably independent of the formation of disulfide bridges, since alkylated polypeptides recovered from gel filtration are dissolved prior to electrophoresis in a solution buffered to pH 6.8 and containing 2% sodium dodecyl sulfate and 5% mercaptoethanol.

Ultrastructure of Whole Fibers (a) Electron microscopy. In all species studied by us, the accessory fibers examined in toto or in section, isolated from or contained in the spermatozoon, exhibit a cross-banding. In the bull the period is clearer (Fig. 6), particularly after negative

300

BACCETTI, PALLINI AND BURRINI T A B L E VI AMINO ACID COMPOSITION OF CEPHALOPOD FIBER POLYPEPTIDE CHAINS (RESIDUES/100 RESIDUES)

Eledone moschata Ve/Vo a MW b MW c A m i n o acid Alanine Arginine A s p a r t i c acid d Cysteine e G l u t a m i c acid ~ Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine

0.9-i.50 5.78 4.04 12.18 3.34 11.81 6.40 1.96 4.83 10.04 6.80 1.13 4.32 6.30 8.00 6.62 " 3.02 3.42

Eledone cirrhose

1.25-1.35 ~95 000 - 9 5 000

1.50-1.60 50 000 53 000

1,83-1.93 28 000 30 000

0.50-1.50 ~ -

1.25-1.42 - 9 0 000 - 9 0 000

1.63-1.78 38 000 36 000

5.75 4.24 9.55 3.37 12.26 6.34 2.55 5.74 7,59 6.78 1.75 3.28 9.37 7.58 5.96 3.05 4,81

4.97 2.58 8.00 9.03 9.96 6.70 6.13 4.11 5.95 4.97 1.23 2,58 14,70 5.66 5.84 1.97 5.56

6.32 2.64 3.95 15.88 6.51 7.80 16.63 3.18 2.63 3.87 0.78 1.17 14.13 6.56 3.80 1.60 2.55

5.09 4.50 9.82 4.96 11.78 7.76 2.44 3.45 7.66 8.02 1.54 2.54 7.65 8.29 6.01 3.54 4.97'

7.73 4.63 10.74 4.85 10.42 5.66 3.18 5.49 8.88 7.11 2.02 3.62 6.25 5.91 5.02 4.13 4.33

6.43 3.48 3.65 15.47 4.24 7,02 17.79 3.98 3.36 2.68 0, 80 0.80 14.60 4.13 3.43 3.04 5.10

a I n d i c a t e s t h e VJVo r a n g e of t h e pooled f r a c t i o n s f r o m gel f i l t r a t i o n (cf. Fig. 5). b M o l e c u l a r w e i g h t f r o m gel f i l t r a t i o n (cf. Figs. 3 a n d 5). M o l e c u l a r w e i g h t f r o m S D S - p o l y a c r y l a m i d e e l e c t r o p h o r e s i s (cf. Fig. 2). a I n c l u d e s a s p a r t i c acid a n d a s p a r a g i n e . e D e t e r m i n e d as S - c a r b o x y m e t h y l c y s t e i n e . i I n c l u d e s g l u t a m i c acid a n d g l u t a m i n e .

staining. It measures 50 nm and is resolved into two areas with different opacity, one containing the first four subbands, and the second five (5). Freeze-etching confirms this picture, and a microdensitometric evaluation (Fig. 7) of photographed PTA preparations reveals a period of 50 nm, clearly divided in two bands, with about nine smaller p e a k s . I n the rat and in the h u m a n the period is not so clear. In rat (Fig. 8) negative staining can reveal only a series of opaque cross-bands 20 nm apart, and this corresponds to the period observed by Woolley (51) and by Fawcett (17) in shadow-casting preparations. Microdensitometric evaluations on our material confirm this measure (Fig. 9). In man, negative staining is unable to reveal periodicity. In thin sections (Fig. 10), as previously pointed out by Pedersen (35), a series of cross-bands less than 20 nm apart can be observed. By microdensitometry (Fig. 11)

on photographed longitudinally sectioned fibers, one 20-nm period is confirmed, with the indication of at least three subbands, but it seems that two consecutive periods can be grouped. But also, in this case, we might have a picture different from that of the bull for the shorter period. In the two species of Eledone examined by us the shadow-casting reveals a clear banding with about a 25-nm period (Fig. 12). After negative staining (Fig. 13), and with the aid of microdensitometric evaluation of the photographs (Fig. 14), a main period some 50 nm long is evident, Consisting of two subbands of different opacity. In each of them several peaks are resolved, giving a picture similar to that observed by Anderson and Personne (2) in gastropods an d by us (3) in Octopus. Probably, two consecutive bands of shadow-casting preparations originate the 50-nm period of negative staining.

50

nm

!

I

@

25

nm

I

I

® 50 I

nm !

® FIG. 6. Bull native accessory fiber. The 50-t~m cross-banding is evident. PTA negative staining. x 130 000. Fro. 7. Microdensitometric scanning of the same. FIG. 8. Rat native accessory fiber. The -20-rim cross-banding is evident. PTA negative staining, x 100 000. FIG. 9. Microdensitometric scanning of the same. FIG. 10. Human native accessory fibers, in longitudinal section. Near the surface the - 2 5 - n m crossbanding is visible, x 140 000. FIa. 11. Microdensitometric scanning of the same. FIG. 12. Eledone cirrhosa native accessory fiber. Shadow casting. The 25-nm cross-superficial banding is evident, x 85 000. Fza. 13. Eledone cirrhosa native accessory fiber. Uranyl formate negative staining. The cross-banding is hardly visible, x 100 000. FIa. 14. Microdensitometric scanning of the same, showing a more evident regular repetition. 301

302

BACCETTI, PALLINI AND BURRINI

Purified fibers digested with papain in the presence of mercaptoethanol, progressively lose their compactness and periodicity and are disrupted. Bull fibers, after a 4-hr treatment, appear to be made up of closely packed filaments (Fig. 15). After 1 or 2 days of enzymic treatment the fasciculate texture of the fibers is more evident, and filaments are frequently isolated and can be easily observed in negative staining. Their diameter is about 2 nm (Fig. 16) and they are not made up of globules. This arrangement is similar to t h a t which sometimes appears in negatively stained untreated fibers (Figs. 17 and 18). When purified bull fibers, reduced in 7 M guanidine-HC1 with 0.1 M dithiothreitol or 0.1 M mercaptoethanol, are dialyzed at 37°C against 0.02 M KC1 containing 3 mM Tris-HC1 buffer of pH 7.5, a precipitate is formed which has mostly an amorphous electron microscopic appearance, but is sometimes organized into fibrous structures. Some of them are simple filaments 2 nm thick (Fig. 19), loosely associated into bundles (Fig. 20); others are more compact fibers (Fig. 21), with the welldefined thickness of 50 nm, still showing the 2 nm longitudinal filaments, laterally packed to give rise to a 25-nm period. Most interestingly, the period of renatured bull fibers is coincident with t h a t of native human and rat fibers. Eledone fibers s h o w , in some cases, a longitudinal filamentous texture in the native state (Fig. 22). The thickness of filaments seems to be about 2 nm, as in the

bull, but they are less evident, After proteolysis the filamentous structure tends to be disrupted, and amorphous clumps are produced as the final stage (Fig. 25). Isolated filaments are never observed, but a filamentous substructure is evident at short proteolysis times (Figs. 23 and 24). (b) Infrared spectra. Figure 26 shows the amide I and amide II regions of the infrared spectrum of bull accessory fibers. Rather well-defined absorption maxima are observed at 1651 cm -1 for the amide I band and 1543 cm -1 for the a~nide II band, which correspond to those characteristic of a-helical proteins (23, 25, 44). The shoulder in the amide I and in the region of 1620-1630 cm -1 may be consistent with an a conformation; the more evident shoulder in the amide II band in the region of 1520-1525 cm -~ may indicate the occurrence of fi or urmrdered conformations. The less evident shoulder in the amide I band at 1660-1665 cm -~ may also be related to an unordered conformation (23,

25, 44). The infrared spectrum of Eledone moschata fibers (Fig. 26) shows a sharp amide I m a x i m u m at 1650 cm -1, consistent with an a helix, with a slight shoulder at 1665 cm -~ and a more evident shoulder at 1630 cm -~, which may be due, respectively, to unordered and fl conformations. The amide II band of Eledone moschata fibers has a maximum at 1535 cm -1 and a shoulder at 1520 cm -~ which may indicate, respectively, fl and unordered conformations

(23, 25, 44).

FIO. 15. Bull accessory fibers proteolyzed to show t h e elementary filamentous structure. Proteolysis conditions: Bull fibers (about 50 mg) were incubated for 6 h r at room t e m p e r a t u r e in 1 ml of a solution containing 0.05 M Tris-HC1 buffer of pH 8.5, 0.1 M mercaptoethanol, and 1 m g of crystalline papain. PTA negative staining, x 100 000. FIG. 16. The same at higher magnification. Two-nanometer-thick filaments are evident. PTA negative staining. × 500 000. Fro. 17. Native gull accessory fibers, showing a compact filamentous structure. U r a n y l formate, x 140 000. FIG. 18. The same at higher magnification. Two-nanometer-thick filaments are evident. U r a n y l formate negative staining. × 650 000. FIG. 19. Renatured filaments from reduced (cf. Materials and Methods) bull accessory fibers. The structures were obtained by dialyzing reduced fiber proteins agains t KC1, 0.02 M containing 3 mM Tris-HC1 buffer of pH 7.5, at 37°C without stirring. PTA negative staining. × 110 000.

FIG. 20. Bundle of filaments obtained by r e n a t u r a t i o n as described above. PTA negative staining. × 80 000. FIG. 21. Fibrous structures showing the elementary filamentous texture and a trace of cross-banding ~arrows), obtained by d e n a t u r a t i o n as described in Fig. 19. PTA negative staining. ×" 110 000. 303

304

BACCETTI, PALLINI AND BURRINI

FIG. 22. Eledone cirrhosa native accessory fiber. PTA negative staining. The filamentous texture (arrow) is visible only in some t h i n n e r region of the fiber, x 200 000. FIG. 23. The same after 30-min proteolysis with mercaptoethanol and papain, as described for Fig. 15. PTA negative staining. A texture of filaments - 2 n m thick is visible (arrows). The most extensive proteolytic degradation (right end of the fiber) originates amorphous clumps, x 200 000. FIG. 24. The same after 2-hr proteolysis. The texture of filaments is e v i d e n t only in some regions (arrows). PTA negative staining, x 200 000. FIG. 25. The same after 24-hr proteolysis. Only amorphous clumps are visible. PTA negative staining, x 100 000.

PARERGINS: PROTEINS OF SPERM ACCESSORY FIBERS

305

90

Bos t a u r u s 70

I 1520

90

~

1543 1625 166.z

r

1651

Eledone

moschata

UJ

O Z <¢ I.I--

70

u~ 150

J

1535

166-'1'/163o / I 1650

30

1800

1600

FREQUENCY

1400

(CM -1)

Fro. 26. Infrared spectra (amide I and amide II regions) of bull and Eledone moschata sperm accessory fibers.

306

BACCETTI, PALLINI AND BURRINI DISCUSSION

The protein constitutions of mammalian and cephalopod accessory fibers have the following common characters: (a) Two classes of polypeptide chains are present. The first has higher molecular weight, relatively lower cysteine and proline and relatively higher leucine and glutamic acid contents. The second has a lower molecular weight, higher cysteine and proline and lower leucine and glutamic acid contents. The chains of the two types are cross-linked by disulfide bridges in the whole fiber. (b) The infrared spectrum (amide I band at 1650 cm -1) indicates the probable presence of an a-helical conformation in both mammals and cephalopods. (c) Different techniques used by us (3, 5) and by other authors (2, 17, 35, 51 ) in the two animal classes substantiate a basic cross-striation with a period of 20-25 nm. Occasionally, two consecutive cross-bands appear grouped, giving a period of - 5 0 nm. In this case several subbands are evident. It is important that this main period has been shown both in mammalian (5) and in gastropod mollusc (2) accessory fibers. (d) Both mammalian and cephalophod fibers contain most of the zinc present in spermatozoa (6). Conceivably it is the polypeptide chains of high cysteine content which bind this metal through free sulfhydryl groups. It is interesting to note that this class of chains in cephalopods also contains a large amount of histidine, whose zinc-binding properties are well known (46). On this basis, the polypeptide chains constituting the accessory fibers may be given the common name of parergins for mammals and molluscs. This class of proteins is quite different from all the other known fibrous proteins except keratins. It is intracellular, and consists of high-sulfur and low-sulfur fractions (10, 21). Nevertheless the peculiar flexibility and localization of the proteins in a wave-generating system allows us to designate them with a specific name. Ex-

periments of proteolysis and renaturation performed on bull parergins clearly provide evidence for 2-nm-thick filaments longitudinally oriented in the fiber. Evidence for filaments of similar thickness is provided by proteolysis and other chemical treatment of keratin, and these filaments are designated as protofibrils (10, 21). Renatured fibers from bull parergin are suggestively similar in their staining characteristics to the fibers isolated from hair root by Whitmore (49). The higher molecular weight polypeptides rich in leucine and glutamic acid (a-helix-forming residues) (20) are conceivably responsible for these protofibril-like structures. In this connection it is interesting to note that the infrared spectrum of bull parergins is rather similar to that of a-keratins (25). Conformations other than an a helix are not to be excluded. The situation is less clear in Eledone parergins, in which the elementary filaments are not evident after proteolysis. In addition, the infrared spectrum of Eledone fibers is less similar to that of a-helical proteins than that of bull fibers. Renatured fibers from bull parergins also suggest that the cross-banding originates from lateral packing of filaments. A cross-striation has also been reported for keratin protofibrils (14) and for keratinlike proteins of the Buccinum egg-capsule wall (39). The low molecular weight class of parergin polypeptides contains cysteine and proline, amino acids which do not favor ordered conformations. These components, therefore, may be regarded as forming an amorphous matrix in analogy with keratin structure (10, 21). This group of chains is probably responsible for the zinc-binding capabilities of the fibers. It has been, in fact, demonstrated that zinc is bound to sulfhydryl groups in mammalian parergins (6, 11, 12, 13). In cephalopod parergins, an aliquot of zinc bound to groups other than sulfhydryls (6) has been detected. This result agrees with the presence of a large amount of histidine in the

PARERGINS: PROTEINS OF SPERM ACCESSORY FIBERS

sulfur-rich chains of cephalopod parergins. Histidine, in fact, forms stable complexes with zinc (46). The interaction between this class of polypeptides and the metal may suggest a mechanism of regulation of the mechanical properties of the fibers, through exchanges of zinc between sperm and seminal fluid (9, 27, 40, 48). Zinc, in fact, influences the degree of cross-linking of sperm proteins (13). The interaction between the low molecular weight chains and zinc may have a further significance in fiber assembly during spermiogenesis. This metal is, in fact, required for correct spermiogenesis (33, 34, 47) and it appears very early in the developing fibers (4, 6). This aspect represents a further similarity between parergins and keratin structures, whose synthesis also requires zinc (38), Moreover, keratohyalin consists of histidine-rich polypeptide chains (43). Histidine is also present in certain mammalian sperm protamines which are eventually cross-linked through disulfides (8, 24). In conclusion, under the name of parergins, the complex of low-sulfur, probably structure-forming, and high-sulfur, probably amorphous, polypeptide chains of the sperm accessory fibers is described. The parergins account for the complete protein fraction of the purified fibers, where no components have been detected with molecular weight and amino acid composition similar to actin or myosin. Attempts to detect dynein chains have also given negative results (unpublished data). The occurrence of ATPase activity in the accessory fibers is still an open question: Only a negligible activity biochemically measured in the purified fibers (5, 36, 37) corresponds to the activity, detected histochemically both in mammals (3, 5, 31) and in molluscs (1, 2, 3). This activity might well be a residue of an extractable ATPase present in the native fiber matrix (52). Our data concerning the protein constitution of the isolated accessory fibers, were not aimed at detecting a contractile activity. The above presented data on parergins

307

support a passive elastic role for the accessory fibers in flagellar wave propagation, in addition to any other function they may or may not have. Such an elastic role agrees with the covalently cross-linked texture of the parergins. In fact, elastin and resilin are covalently cross-linked proteins, and keratin (wool) has well-known elastic properties (21). It is interesting to recall that the mechanical properties of wool depend on thiol-disulfide exchanges; see Torchinskii (45) for a discussion. The relatively high number of thiol groups of parergins (6) is suggestive of a similar reaction in accessory fibers.

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